Creation of Embryoid Bodies from iPSCs using Complete KnockOut™ Serum Replacement Feeder-Free Medium

• Sterile Tissue Culture Hood
• Incubator set at 37°C
• Pipette-Aid
• Water Bath set at 37°C
• Sterile serological pipettes (5 mL, 10 mL)
• 15 mL centrifuge tubes
• 60 mm Tissue Culture treated dishes
• 60 mm or 100 mm non tissue culture treated dishes

1. To prepare 100 mL of Complete KnockOut™ Serum Replacement EB medium, aseptically combine the components listed in the table below.
   
   

   Component	Stock Concentration	Final Concentration	Volume
   Knockout™ DMEM/F12 (Cat. no. 12660-012)	-	1X	78 mL
   GlutaMAX™ -I (Cat. No. 35050-061)	200 mM	2 mM	1 mL
   KnockOut™ SR (Cat. no. 10828-028)	-	20%	20 mL
   Non-Essential Amino Acids (Cat. no. 11140-076)	10 mM	0.1 mM	1

   
   Note: The KSR EB medium can be prepared and stored at 2-8°C for up to 2 weeks.
   
   
2. Just before pre-equilibrating the complete medium to temperature and gases, aseptically add the required volume of 2-mercaptoethanol (55 mM stock concentration) for a 0.1 mM final concentration. For example, to prepare 100 mL of KSR-FF medium add 182 μL of 55 mM 2-mercaptoethanol (1:550 dilution) Alternatively, the 2-mercaptoethanol may be added to the 1X completed medium and stored at 2–8°C for up to one week.
3. Aliquot appropriate volume (see Table 1) of KSR EB Medium needed for the day to a centrifuge tube warm to 37°C.
   

1. Observe the human iPSCs growing in complete KSR-Feeder Free medium under the microscope to confirm that the cells are 70–80% confluent and ready to be subcultured.
   
   Note: If colonies become too dense or too large, increased differentiation occurs.
   
   
2. Cut out and remove any differentiated iPSC or hESC colonies prior to passaging the culture.
3. Pre-warm the required volume of Dispase in a 37 °C water bath. Refer to Table 1 below for details on the volumes required.
4. Pre-equilibrate the required volume of KSR-EB medium in a 37°C water bath for15 min. Refer to Table 1 below for details on the volumes required.
5. Aspirate the spent medium from the culture dish using a pipette, and rinse the cells twice with D-PBS.
6. Gently add pre-warmed Dispase solution to the culture dish (e.g., 1 mL of Dispase solution per 60-mm culture dish). Swirl the culture dish to coat the entire cell surface.
7. Incubate the culture dish at 37°C for 3 minutes.
8. Remove the dish from the incubator, aspirate the Dispase solution, and gently wash the cells with D-PBS.
9. Gently scrape the cells off the surface of the culture dish using a cell scraper, and transfer the cells to a sterile 15 mL centrifuge tube.
10. Rinse the culture dish twice with DPBS, gently “spraying off” any cells that have not detached. Pool the rinse with the cells in the 15 mL tube.
11. Centrifuge the tube at 200 × g for 5 minutes at room temperature to pellet the cells.
12. Carefully aspirate the supernatant without disturbing the cell pellet and discard it.
13. Gently flick the tube to fully dislodge the cell pellet from the tube bottom.
14. Gently resuspend the cells in pre-equilibrated complete KSR-EB medium using a 5-mL serological pipette. Do not triturate. Note: it is critical at the step to gently resuspend the cells without using force to avoid damage.
15. One 60 mm dish of cells can be transferred to one 60mm EB dish or one 100 mm EB dish. Place the cells onto a 60 mm or a 100 mm non tissue culture treated dish.
16. Place the culture dish in a 37°C incubator with a humidified atmosphere of 4 to 6% CO₂ in air.
17. Change the medium on the EBs every other day by pulling off the entire volume of the dish and transferring it into a centrifuge tube. Keep the tube in the hood and allow the cells to settle to the bottom of the tube (about 5 minutes). Then, using a pipette, remove the supernatant from the tube and replace it with fresh, equilibrated KSR EB medium. Place the cells back onto the same dish.
18. Continue to change the medium every other day. The EBs will grow in size over time.
19. Alternatively, after 4 days, transfer the cells equally into 2 x 100mm tissue culture treated dishes to allow for attachment. The attachment of EBs will allow for further differentiation of the cells into various cell types.
20. Keep the same schedule for fluid changes over time, however as the cells expand the volume per dish may need to be increased by 50% or 100%.
21. Allow the cells to expand for time points at 14 days and 21 days, or even longer. The entire EB dish can be harvested for analysis.

Figure 1: Embryoid bodies at various time points (Day 3, 7, 9, and 16), all at 40 x magnification. Spheres gradually turn into masses of cells, and once transferred onto tissue culture treated dishes, they eventually attach and differentiate.

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